Gastrointestinal infections claim the lives of 2-3 million children annually. Interim data from a Global Enteric Multicenter Study indicate that a disporportionate number of deaths among children is associated with infections due to enteropathogenic E. coli (EPEC) and Shigella spp. In this project we will investigate the contribution to pathogenesis of specific factors produced by EPEC and Shigella that are associated with severe disease and we will discover new such factors in EPEC. We will determine the genome sequence of 20 EPEC strains recently isolated from children who succumbed to diarrhea, 20 strains from children with non-lethal sypmptomatic infection and 20 strains from asymptomatic control children. We will specifically seek new genetic loci that are associated with symptomatic infection and with lethal outcome. We will determine the host cell targets and mechanisms of action of two proteins, NIeB and NIeE, that are secreted by EPEC and injected into host cells and that have been associated in previous studies with increased virulence in humans. We will also determine the contributions to pathgenesis of novel EPEC genetic loci associated with virulence identified by the above genomic sequencing. And we will determine the target and mechanism of action of a protein known as ShET2 that is produced and secreted by both EPEC and Shigella, associated with increased virulence in human EPEC infection, and has been proposed but not proven to be injected into host cells. Together, these experiments will yield an unprecidented amount of data on the genetic basis of EPEC pathogenesis, uncover mechanisms of action of virulence factors from EPEC and Shigella, and in particular identify novel loci associated with deadly infections. These data will serve as an extremely valuable resource for new initiatives to prevent and treat severe enteric infections.